Chronic graft-vs.-host disease (GVHD) is the major morbidity and mortality of long-term survivors of allogeneic hematopoietic cell transplantation (HCT), and therapies that prevent acute GVHD are unsuccessful in preventing chronic GVHD. Chronic GVHD is considered an autoimmune collagen-vascular disease with clinical features similar to autoimmune scleroderma and systemic lupus erythematosus (SLE). However, the pathogenesis of chronic GVHD is poorly understood. It is unclear how autoreactive T cells are generated in chronic GVHD recipients; it is largely unknown how B cells and autoantibodies contribute to the pathogenesis, although it was recently reported that anti-CD20 mAb ameliorated refractory chronic GVHD by depleting B cells. We recently developed a new model of chronic GVHD, in which DBA/2 donor (H-2d) spleen cells were injected into sub-lethally irradiated MHC matched but minor antigen mismatched BALB/c (H-2d) recipients, and the recipients developed autoimmune-like GVHD with high levels of serum autoantibodies, sclerodermatous skin damage, and glomerulonephritis. Disease induction required both donor CD4+ T and B220+ B cells in transplants. In contrast, addition of donor CD25+CD4+ regulatory T [unreadable] (Treg) cells to transplants prevented the disease development. Therefore, we hypothesize that activation and expansion of the quiescent donor autoreactive CD4+ T and B cells from transplants in allogeneic recipients lead to the development of chronic GVHD. Furthermore, the activated autoreactive B cells play a central role in the activation of autoreactive CD4+ T cells and amplification of the autoimmune response. In contrast, Treg cells suppress the autoimmune response in chronic GVHD. To test our hypothesis, we will 1) determine the origin of autoreactive CD4+ T cells by comparing disease induction in euthymic and athymic recipients; and identify the donor CD4+T cell subsets in transplants responsible for the disease induction; 2) determine the role of donor autoreactive B cells in transplants in the activation of autoreactive CD4+ T cells and the disease induction; 3) determine whether natural as well as Foxp3 transduced CD25+CD4+Treg cells can be used to prevent and treat autoimmune-like chronic GVHD. These studies will provide new insights into the pathogenesis of chronic GVHD, and provide new approaches for preventing and treating chronic GVHD. [unreadable] [unreadable] [unreadable]